The Experts below are selected from a list of 8754 Experts worldwide ranked by ideXlab platform
Nathaniel Heintz - One of the best experts on this subject based on the ideXlab platform.
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rapid bacterial artificial Chromosome Modification for large scale mouse transgenesis
Nature Protocols, 2010Co-Authors: Shiaoching Gong, Laura Kus, Nathaniel HeintzAbstract:We report here a high-throughput method for the Modification of bacterial artificial Chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.
Shiaoching Gong - One of the best experts on this subject based on the ideXlab platform.
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rapid bacterial artificial Chromosome Modification for large scale mouse transgenesis
Nature Protocols, 2010Co-Authors: Shiaoching Gong, Laura Kus, Nathaniel HeintzAbstract:We report here a high-throughput method for the Modification of bacterial artificial Chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.
Laura Kus - One of the best experts on this subject based on the ideXlab platform.
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rapid bacterial artificial Chromosome Modification for large scale mouse transgenesis
Nature Protocols, 2010Co-Authors: Shiaoching Gong, Laura Kus, Nathaniel HeintzAbstract:We report here a high-throughput method for the Modification of bacterial artificial Chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.
Ohbayashi Tetsuya - One of the best experts on this subject based on the ideXlab platform.
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Application of a Bacterial Artificial Chromosome Modification System for a Human Artificial Chromosome Vector
'Tottori University Faculty of Medicine', 2020Co-Authors: Yamaguchi Shigeyuki, Niwa Ryosuke, Kazuki Yasuhiro, Ohbayashi TetsuyaAbstract:Exactly controlled conditional gene expressing systems are crucial for genomic functional research, animal transgenesis and gene therapy. Bacterial artificial Chromosomes (BACs) are optimal for harboring long fragments of genomic DNA or large cDNA up to 300 kb in size. Therefore, BACs are available to produce transgenic cells and animals for the functional studies of genes. However, BAC can insert DNA randomly into the host genome, possibly causing unpredicted expression. We previously developed a human artificial Chromosome (HAC) vector from human Chromosome 21 using Chromosome engineering. The HAC vector has several important characteristics desired for an ideal gene delivery vector, including stable episomal maintenance, and the ability to carry large genomic DNA containing its own regulatory element, thus allowing physiological regulation of the transgene in a manner similar to that of the native Chromosome. In this study, we develop a system fusing BAC library and HAC technology together to allow tight control of gene expression. This system enables BAC to be cloned into the defined locus on the HAC vector by the Cre/loxP system. In addition, the genome in the BAC is possible to be engineered freely by the BAC recombineering technology. This system is a highly efficient tool for the rapid generation of stringently controlled gene expression system on the HAC vector
Mitsuo Oshimura - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 induced transgene insertion and telomere associated truncation of a single human Chromosome for Chromosome engineering in cho and a9 cells
Scientific Reports, 2017Co-Authors: Narumi Uno, Kei Hiramatsu, Shinya Komoto, Yasuhiro Kazuki, Katsuhiro Uno, Mitsuo OshimuraAbstract:Chromosome engineering techniques including gene insertion, telomere-associated truncation and microcell-mediated Chromosome transfer (MMCT) are powerful tools for generation of humanised model animal, containing megabase-sized genomic fragments. However, these techniques require two cell lines: homologous recombination (HR)-proficient DT40 cells for Chromosome Modification, and CHO cells for transfer to recipient cells. Here we show an improved technique using a combination of CRISPR/Cas9-induced HR in CHO and mouse A9 cells without DT40 cells following MMCT to recipient cells. Transgene insertion was performed in CHO cells with the insertion of enhanced green fluorescence protein (EGFP) using CRISPR/Cas9 and a circular targeting vector containing two 3 kb HR arms. Telomere-associated truncation was performed in CHO cells using CRISPR/Cas9 and a linearised truncation vector containing a single 7 kb HR arm at the 5′ end, a 1 kb artificial telomere at the 3′ end. At least 11% and 6% of the targeting efficiency were achieved for transgene insertion and telomere-associated truncation, respectively. The transgene insertion was also confirmed in A9 cells (29%). The modified Chromosomes were transferrable to other cells. Thus, this CHO and A9 cell-mediated Chromosome engineering using the CRISPR/Cas9 for direct transfer of the modified Chromosome is a rapid technique that will facilitate Chromosome manipulation.